Research Association of circulating angiotensin converting enzyme

Dimitriou et al. Respiratory Research 2010, 11:57 http://respiratory-research.com/content/11/1/57

Open Access

RESEARCH

Association of circulating angiotensin converting enzyme activity with respiratory muscle function in infants Research

Gabriel Dimitriou*1, Despina Papakonstantinou1, Eleana F Stavrou2, Sotirios Tzifas1, Aggeliki Vervenioti1, Anny Onufriou3, Aglaia Athanassiadou2 and Stefanos Mantagos1

Abstract Background: Angiotensin converting enzyme (ACE) gene contains a polymorphism, consisting of either the presence (I) or absence (D) of a 287 base pair fragment. Deletion (D) is associated with increased circulating ACE (cACE) activity. It has been suggested that the D-allele of ACE genotype is associated with power-oriented performance and that cACE activity is correlated with muscle strength. Respiratory muscle function may be similarly influenced. Respiratory muscle strength in infants can be assessed specifically by measurement of the maximum inspiratory pressure during crying (Pimax). Pressure-time index of the respiratory muscles (PTImus) is a non-invasive method, which assesses the load to capacity ratio of the respiratory muscles. The objective of this study was to determine whether increased cACE activity in infants could be related to greater respiratory muscle strength and to investigate the potential association of cACE with PTImus measurements as well as the association of ACE genotypes with cACE activity and respiratory muscle strength in this population. Methods: Serum ACE activity was assayed by using a UV-kinetic method. ACE genotyping was performed by polymerase chain reaction amplification, using DNA from peripheral blood. PTImus was calculated as (Pimean/Pimax) × (Ti/Ttot), where Pimean was the mean inspiratory pressure estimated from airway pressure, generated 100 milliseconds after an occlusion (P0.1), Pimax was the maximum inspiratory pressure and Ti/Ttot was the ratio of the inspiratory time to the total respiratory cycle time. Pimax was the largest pressure generated during brief airway occlusions performed at the end of a spontaneous crying effort. Results: A hundred and ten infants were studied. Infants with D/D genotype had significantly higher serum ACE activity than infants with I/I or I/D genotypes. cACE activity was significantly related to Pimax and inversely related to PTImus. No association between ACE genotypes and Pdimax measurements was found. Conclusions: These results suggest that a relation in cACE activity and respiratory muscle function may exist in infants. In addition, an association between ACE genotypes and cACE activity, but not respiratory muscle strength, was demonstrated.

Background Angiotensin I-converting enzyme (ACE) is a zink metallopeptidase whose main functions are to convert angiotensin I into vasoactive and aldosterone-stimulating peptide angiotensin II and to degrade vasodilator kinins. * Correspondence: [email protected] 1

Neonatal Intensive Care Unit, Department of Pediatrics, University of Patras Medical School, Rio, Patras, Greece

Full list of author information is available at the end of the article

Circulating ACE (cACE) is found in biological fluids and originates from endothelial cells. ACE is also an important component of the local renin-angiotensin systems (RASs), which have been identified in diverse tissues, including lung and skeletal muscles [1,2]. A polymorphism of the human ACE gene has been identified in humans and contains a polymorphism consisting of either the presence (insertion, I) or absence (deletion, D) of a 287 base pair (bp) fragment [3]. The deletion is asso-

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ciated with increased ACE activity in both tissue [4] and circulation [5]. Circulating ACE activity was stable when serially measured in the same individuals, while large differences among subjects were observed [6]. The I/D polymorphism accounts for approximately half of the observed variance in ACE levels [5]. However, the presence of quantitative trait loci controlling ACE levels was suggested [7]. D-allele of ACE genotype has been associated with power-oriented performance, being found in excess in short-distance swimmers [8] and with greater strength gains in the quadriceps muscle [9]. Furthermore, it has been suggested that cACE activity has been associated directly with muscle strength in healthy Caucasians, naïve to strength training [10]. Thus, respiratory muscle function and specific respiratory muscle strength may be similarly influenced. Respiratory muscle strength in infants can be assessed specifically by measurement of the maximum inspiratory pressure during crying (Pimax) [11,12]. Pressure-time index of the respiratory muscles (PTImus) is a non-invasive method, which assess the load to capacity ratio of the respiratory muscles [13]. PTImus has been validated in both adults [14] and infants [15]. The aim of this study was to test the hypothesis that increased cACE activity in infants could be related to greater respiratory muscle strength assessed by measurement of Pimax. We further investigated the potential association of cACE with PTImus measurements, as well as the association of ACE genotypes with cACE activity and respiratory muscle strength in this population.

Methods Patients

Infants cared for at the Neonatal Intensive Care UnitPediatric Department of the University General Hospital of Patras, Greece, were eligible for the study. Infants were entered into the study if parents gave informed written consent. The study was approved by the local Research Ethics Committee. The studied population was recruited from a study examining the association of ACE genotypes on respiratory muscle function in infants. All infants were studied before discharge, in supine position, at least one hour after a feed. Infants had no respiratory symptoms for at least 3 days before measurement. Furthermore, infants were on full oral feeds, had serum electrolytes, calcium, magnesium and phosphates within normal range and did not receive any methylxanthines. Blood sampling for circulating ACE activity determination was performed the previous or the same day of the measurements. ACE genotype determination

ACE genotyping was performed on DNA extracted from 0.5 ml of whole blood, collected from an indwelling cath-

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eter or via peripheral venipuncture during routine blood sampling. The blood samples were stored at -80°C in EDTA vacutainer tubes. The method has been previously described [16]. Briefly, DNA was extracted by using Qiamp spin columns (Blood mini kit- Qiagen, QIAGEN Inc., Germantown, U.S.A). DNA was analyzed by electrophoresis on an agarose gel. DNA amplification of the 16th ACE intron was performed using two sets of primers flanking the polymorphic site, (outer and inner primers), as mistyping of the D/D genotype has been reported to occur using conventional amplification with insertion/ deletion (I/D) flanking primer [17]. Plasma ACE activity determination

An additional 1 ml of whole blood was collected during routine blood sampling. Serum was separated immediately from the whole blood by centrifugation at 1500 g for 10 min. The samples were stored at -20°C in vacutainer tubes until analysis. Serum ACE activity was assayed by using a UV-kinetic method (Medicon SA) and an AU480 Clinical Chemistry System (Beckman Coulter, Inc, High Wycombe, UK). The determination of ACE was based on the calculation of the rate of absorbance change at 340 nm during the hydrolysis of the substrate N-(3-(2(furyl)acryloyl)-L-phenylalanylglycylglycine (FAPGG) to N-(3-(2-(furyl)acryloyl)-L-phenylalanine (FAP) and glycylglycine. The method has a detection limit of 7 U/L, linearity between 7-140 U/L of ACE and an intra-assay coefficient of variation between 2.26% and 4.86%. Measurement of respiratory muscle function

Airway flow was measured using a pneumotachograph (Mercury F10L, GM Instruments, Kilwinning, Scotland) connected to a differential pressure transducer (DP45, range ± 2 cm H2O, Validyne Corp, Northridge, CA, USA). Airway pressure (Paw) was measured from a side port on the pneumotachograph, using a differential pressure transducer (DP45, range ± 100 cm H2O, Validyne Corp, Northridge, CA, USA). The signals from the differential pressure transducers were amplified, using a carrier amplifier (Validyne CD 280, Validyne Corp, Northridge, CA, USA) and they were recorded and displayed in real time on a computer (Dell Optiplex GX620, Dell Inc., Texas, U.S.A) running Labview™ software (National Instruments, Austin, Texas, U.S.A) with analog-to-digital sampling at 100 Hz (16-bit NI PCI-6036E, National Instruments, Austin, Texas, U.S.A). Measurement of Pimax

To measure Pimax, a facemask (total deadspace, 4.5 mL) was held firmly over the infant's nose and mouth. A small needle leak in the mask was used in order to prevent glottic closure and artificially high Pimax [13]. The airway was occluded at the end of a spontaneous crying effort using a unidirectional valve attached to the pneumotachograph, which allowed expiration but not inspiration. The occlu-


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Table 1: Characteristics of the whole study population Number of studied infants

110

Gestational age (weeks)

36

Birth weight (g)

Table 1: Characteristics of the whole study population Pimax (cmH2O)

65.0

(36.9-91.8)

(27-40)

Pimax/weight (cmH2O/kg)

24.5

(14.5-47.1)

2705

(960-4150)

PTImus

0.062

(0.023-0.149)

Gender (male)

62

(56.4)

Preterm

75

(57.7)

Data are demonstrated as n (%) or median (range). Abberviations: cACE: circulating ACE; Pimax: maximum inspiratory pressure during crying; PTImus: pressure-time index of the respiratory muscles

Duration of mechanical ventilation (days)

0

(0-59)

Respiratory distress syndrome (RDS)

37

(33.6)

Transient tachypnea of the newborn (TTN)

17

(15.5)

Meconium aspiration syndrome

5

(4.5)

Infection

16

(14.5)

Congenital pneumonia

6

(5.5)

Prematurity (gestational age ≤ 34 weeks and birthweight < 2 kg without additional problems on admission)

15

(13.7)

Birth depression

6

(5.5)

Intrauterine growth retardation

5

(4.5)

P0.1 was calculated as the airway pressure generated 100 milliseconds after an occlusion, while the infant was quietly breathing. At least four airway occlusions were performed and average P0.1 was calculated. Pressure-time index of the inspiratory muscles (PTImus), was calculated as: PTImus = (Pimean/Pimax) × (Ti/Ttot) where Pimean was the average airway pressure during inspiration, obtained from the formula Pimean = 5 × P0.1 × Ti [18]. Pimax was the maximum inspiratory airway pressure, Ti was the inspiration time and Ttot was the total time for each breath, calculated from the airway flow signal. Muscle mass increases with maturity and body growth [19] and Pimax continues to increase outside the neonatal period [11]. Therefore, in order to examine the association of ACE genotype with respiratory muscle strength, Pimax was also related to body weigth at the time of measurement.

Airleaks

2

(1.8)

Statistical analysis

Diagnoses on admission

sion was maintained for at least four inspiratory efforts. At least three sets of airway occlusions were performed and the maximum Pimax achieved for individual was recorded. Measurement of PTImus

Meconium plug

1

(0.9)

Postnatal age (days)

9

(1-107)

Postmenstrual age (PMA) (weeks)

37.1

(32.4-46.7)

Preterm (PMA